Our goal is development of a multiplex immunodiagnostic panel specific for Giardia, Cryptosporidium, E. histolytica, and shiga toxin-producing Shigella and E. coli detection in stool. Additional effort will quantify the fecal biomarkers lactoferrin and hemoglobin, indicators of intestinal inflammation and hemorrhage. The full multiplex panel will provide rapid diagnosis of major Category B enteropathogenic agents of diarrhea/dysentery and describe severity of disease. The assay utilizes sensitive and specific immunofluorescence-based Luminex technology. The Luminex allows multiplexed detection of analytes in a single sample using an array of uniquely detectable bead sets. Analyte-specific antibodies are selected from existing FDA-cleared immunoassays, providing assurance of their performance and stability. Preliminary data indicate Luminex sensitivity for the enteric protozoa exceeds that of ELISA, and is comparable to RT-PCR, with purified parasite curves. Detection in stool identified 108 of 108 parasite FDA-cleared ELISA positive reactions, but still requires complete validation of clinical sensitivity. Luminex detection of lactoferrin and hemoglobin correlate with commercial ELISA detection and provide an expanded range of quantification, increasing efficiency of fecal analysis over ELISA. Aim #1 details an enteric protozoan parasite panel for Giardia, Cryptosporidium, and E. histolytica. Aim #2 expands this diarrhea/dysentery panel to include detection of shiga toxin, shiga-like toxin (Stx1) and shiga-like toxin 2 (Stx2). Aim #3 adds detection of lactoferrin and hemoglobin as biomarkers of diarrhea/dysentery severity and patient condition. Successful completion of these studies will provide a prototype diagnostic capable of the simultaneous detection of Category B enteropathogens and intestinal biomarkers that describe severity of infection. The multiplex assay will be competitive for Phase II NIH small business funding to complete manufacturing transfer and FDA 510(k) clearance validations. The Luminex-based multiplex detection is an excellent candidate for commercialization, and it allows for future inclusion of other enteric pathogens and gut function biomarkers. PUBLIC HEALTH RELEVANCE: This study proposes a diagnostic panel to identify the Category B Biodefense intestinal pathogens Giardia, Cryptosporidium, E. histolytica, and shiga toxin-producing Shigella and E. coli in patients with diarrhea and dysentery. The panel will also quantify fecal biomarkers to describe intestinal inflammation and hemorrhage, providing novel information regarding disease severity and patient condition.